1. Field of the Invention
The present invention relates to genetically engineered baculoviruses substantially incapable of producing the baculovirus LEF-8 RNA polymerase subunit and eukaryotic cells infected with such baculoviruses. Such baculoviruses comprise an engineered chromosome in which the lef-8 gene has been inactivated or deleted. The invention is also related to methods for expressing heterologous proteins in cells using such baculovirus artificial chromosomes.
2. Description of the Related Art
The insect order of Lepidoptera includes all butterflies and moths, such as the silkmoth Bombyx mori and the alfalfa looper Autographa californica. Lepidopteran insects are host organisms for some members of a family of viruses, known as baculoviruses (more than 400 known species), that infect a variety of arthropods. In nature a baculovirus enters a lepidopteran insect host when a proteinaceous structure containing the virus, known as an "occlusion body" or "polyhedrin", is ingested with food. Once the occlusion body is dissolved in the insect gut, freed virus particles enter the host cells. This is followed by replication of viral genomic DNA, and by the generation and release of viral progeny for infection of other host insect tissues. The infection process terminates with the formation of occluded viruses prior to host cell death.
After the insect dies from infection, occlusion bodies containing virus are released from the dead larvae into the environment and spread the infection to other insects through contamination of the food supply. These occlusion bodies serve to protect the virus particles in the environment and also provide a means of delivering the virus particles to the primary site of infection in insects, the midgut epithelial cells.
Secondary infection within the insect involves the budded form of the virus. Viral nucleocapsids are synthesized in the nucleus of the insect cell, move through the cytoplasm and bud from the plasma membrane of the cell resulting in the release of budded virus particles into the insect hemolymph. The open circulatory system of the insect provides the virus with access to other tissues of the insect.
The synthesis of the budded and occluded forms of the virus is temporally regulated. During a typical infection of host tissue culture cells, progeny budded viruses are released into the culture media beginning approximately 12 hours post infection (p.i.) and the release continues logarithmically through 22 hours p.i. Occluded virus forms approximately at 20 hours p.i. and continues through 70 hours p.i. by which time approximately 70-100 polyhedral occlusions have formed in the nucleus. This temporal regulation of viral development is reflected in the controlled transcription of specific viral genes.
Baculoviruses, specifically nuclear polyhedrosis viruses (NPVs) have a unique life cycle which involves the temporally regulated expression of different genes. Nuclear polyhedrosis virus genes are transcribed in a regulated cascade involving at least three phases of transcription: an early phase (0-6 hours p.i.) prior to viral DNA replication, a late phase (6-18 hours p.i.) involving DNA replication and budded virus formation and the very late occlusion phase (18 through 70 hours p.i.). See FIG. 1.
Hashimoto et al., (1996), "Attenuation of Bombyx mori nuclear polyhedrosis for a gene transfer vector", p70 in Japan-Canada Cooperative Workshop disclosed a temperature sensitive mutant Bombyx mori NPV which underwent DNA replication but did not produce extracellular virus and polyhedra. Hashimoto indicated that the mutations were within a genomic region of 7.3 mu to 32.5 mu which contained the p47, p39 and lef11 AcNPV gene homologues.
To date, the study of lepidopteran insects and commercial biotechnology applications using lepidopteran insects or in vitro cell cultures derived from such insects, has been hampered by the lack of a suitable method to permanently introduce genes into the host (a stable gene transformation or transduction system).
In contrast, successful gene transformation systems have been developed for non-lepidopteran insect species, such as the fruit fly Drosophila melanogaster, where P element-based embryo transformation vectors have led to an explosion in the understanding of the complex genetic processes involved in the fruit fly life cycle.
A novel stable gene transduction (or transformation) methodology that allows the generation of (1) transgenic lepidopteran insects that can be used for basic research, recombinant protein production and biopesticide industry-related applications, (2) in vitro transduced lepidopteran insect cell cultures for large scale production of recombinant proteins would be advantageous, and (3) in vivo DNA vaccination and gene therapy would be desirable.
It has now been found that the inactivation or deletion of the lef-8 gene from the baculovirus genome allows the baculovirus genome to replicate but not to kill the insect cell or form occlusion bodies. Thus the baculovirus artificial chromosome cannot productively infect insect cells. Such recombinant baculovirus genomes can function as artificial chromosomes for the production of recombinant proteins in insect cells.
The baculovirus genome encodes its own RNA polymerase enzyme which is composed of a number of different subunits. One of the baculovirus genes encoding a RNA polymerase-like subunit, LEF-8, of the Bombyx mori nuclear polyhedrosis virus (BMNPV) genome has been identified and sequenced (Genbank Accession Number L33180:ORF #39). This gene is conserved among all the characterized baculoviruses. For example, the equivalent gene in Autographa californica (AcMNPV), encoded by open reading frame (ORF) #50, has also been sequenced (GenBank Accession number L22858), as has the gene of Orgyia pseudotsugata (OpMNPV), encoded by ORF #54 (GenBank Accession Number U75930).
Further advantages of the present invention will become apparent from the following description of the invention with reference to the attached drawings.